A technology to induce differentiation of pluripotent stem cells holds the key for realization of regenerative medicine and establishment of in vitro evaluation of drug efficacy and safety. In particular, the regenerative medicine and drug evaluation for heart diseases, which are the second cause of death in Japan, are important. Additionally, stable provision of homogenous cardiomyocytes is needed for cardiotoxicity study of various drugs since many drugs induce severe cardiac side effects including cardiac arrest and arrhythmia.
It has been reported that cardiac muscle differentiation of human ES/iPS cells is induced by co-culturing human ES cells and mouse feeder cells, END2 cells (Non Patent Literature 1, the reference is incorporated herein by reference). Unfortunately, the differentiation efficiency is low and the resulting human cardiomyocytes are not pure because of contamination of the mouse END2 cells.
It is also reported that cardiac muscle differentiation of human ES/iPS cells is induced by preparing embryoid from ES cells and adding several cytokines (fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), vascular endothelial cell growth factor, DKK1, Activin A) to the embryoid (Non Patent Literatures 2 and 3, the references are incorporated herein by references). It is also reported that cardiac muscle differentiation is induced with BMP4, FGF2, insulin and serum (Non Patent Literature 4, the reference is incorporated herein by reference). These methods, however, are not suitable for practical use due to need of huge amount of cytokines which increases cost. It is also reported that cardiac muscle differentiation of mouse ES cells is induced with XAV939, a tankyrase inhibitor (Non Patent Literature 5, the reference is incorporated herein by reference). Unfortunately, it is difficult to use the resulting cells for regenerative medicine since serum is used in the preparation of the cells and the differentiation efficiency is low as 10 to 60%.
The inventors of the present invention have reported low molecular weight compounds that promote cardiac differentiation of pluripotent stem cells (Patent Literatures 1 and 2, and Non Patent Literature 7, the references are incorporated herein by references).
Epidermal growth factor (EGF) receptor (EGFR) is a receptor tyrosine kinase, the ligand of which is EGF. EGFR is important for regulating proliferation, differentiation and maintenance of cells. EGF signaling has been reported to play an important role in development of heart (Non Patent Literature 6, the reference is incorporated herein by reference). EGFR is also involved in proliferation and metastasis of cancer cells. EGFR inhibitors such as gefitinib are used as an anti-cancer agent. However, use of EGFR inhibitors such as gefitinib and AG1478 has not been reported for inducing cardiac differentiation of human pluripotent stem cells.